A nitrilase-mediated entry to 4-carboxymethyl-β-lactams ... · 1 Electronic Supplementary...

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  • 1

    Electronic Supplementary Information

    A nitrilase-mediated entry to 4-carboxymethyl-β-lactams from

    chemically prepared 4-(cyanomethyl)azetidin-2-ones

    Lena Decuyper,a Nicola Piens,a Jens Mincke,a Jeroen Bomon,a Bert De Schrijver,a Karen Mollet,a Karel

    De Winter,b Tom Desmetb and Matthias D’hooghe*a

    aSynBioC Research Group, Department of Sustainable Organic Chemistry and Technology, Faculty of

    Bioscience Engineering, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. E-mail:

    [email protected]

    bDepartment of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent

    University, Coupure Links 653, B-9000 Ghent, Belgium

    Contents

    Experimental part with procedures and characterization data of compounds 6a,c, 7a-c, 8a,b, 9a-c,

    10a-c, 11a-c, 12a-c 2 – 11

    1H NMR and 13C NMR spectra of compounds 6a,c, 7a-c, 8a,b, 9a-c, 10a-c, 11a-c, 12a-c 12 – 49

    Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2016

  • 2

    Experimental part

    General methods

    Commercially available solvents and reagents were purchased from common chemical suppliers and

    used without further purification. Melting points were measured using a Kofler bench, type WME

    Heizbank of Wagner & Munz. Optical rotations were determined with an JASCO P-2000 series

    polarimeter. Elemental analyses were obtained by means of a Perkin-Elmer 2400 Series II apparatus.

    IR spectra were obtained from samples in neat form with an ATR (Attenuated Total Reflectance)

    accessory with a Perkin-Elmer Spectrum BX FT-IR spectrophotometer. 1H NMR spectra were recorded

    at 300 MHz (Jeol Eclipse+ 300) or at 400 MHz (Bruker Avance III-400) in deuterated solvents with

    TMS as internal standard. 13C NMR spectra were recorded at 75 MHz (Jeol Eclipse+ 300) or at 100

    MHz (Bruker Avance III-400). Electron spray (ES) mass spectra were obtained with an Agilent 1100

    Series MS (ES, 4000V) mass spectrometer. High resolution electron spray (ES-TOF) mass spectra were

    obtained with an Agilent Technologies 6210 Series Time-of Flight.

    Synthesis of 4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-ones 6a–c

    4-[(4S)-2,2-Dimethyl-1,3-dioxolan-4-yl]azetidin-2-ones 6a–c were prepared according to a literature

    procedure.45,47,48

    (3R,4S)-1-Isopropyl-3-methoxy-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-one 6a

    Yellow oil; yield: 9.00 g (85%); Rf 0.13 (hexane/EtOAc 4/1); [α]D +37.2 (c 0.33 in CHCl3); IR (νmax/cm-1):

    1747 (C=O), 2982, 2936, 1370, 1211, 1064, 1034, 851; 1H NMR (400 MHz, CDCl3): δ 1.28 (6H, d, J = 6.8

    Hz, NCH(CH3)2), 1.35 and 1,45 (2 x 3H, 2 x s, Cquat(CH3)2),

    3.53 (3H, s, OCH3), 3.59 (1H, d x d, J = 7.9, 5.5

    Hz, O(HCH)), 3.68 (1H, d x d, J = 8.7, 5.2 Hz, OCHCHN), 3.91 (1H, septet, J = 6.8 Hz, NCH(CH3)2), 4.13-

    4.22 (2H, m, O(HCH)CH), 4.35 (1H, d, J = 5.2 Hz, CH3OCH); 13C NMR (100 MHz, CDCl3): δ 19.4 and 21.3

    (NCH(CH3)2), 25.1 and 26.8 (Cquat(CH3)2), 44.3 (NCH(CH3)2), 59.1 (OCH3), 59.8 (OCHCHN), 66.9 (OCH2),

    77.0 (CH2CHO), 82.0 (CH3OCH), 109.3 (Cquat(CH3)2), 166.9 (C=O); MS (70 eV): m/z (%) = 244 (M+ + H,

    100); HRMS (ESI): m/z [M + H]+ calcd for C12H22NO4: 244.1549, found: 244.1550.

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-one 6b

    Spectral data were in accordance with those reported in the literature.42

    (3R,4S)-1-Butyl-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-phenoxyazetidin-2-one 6c

    White crystals; yield: 2.84 g (89%); Rf 0.24 (hexane/EtOAc 4/1); mp 52 °C; [α]D +294.8 (c 0.33 in

    CHCl3); IR (νmax/cm-1): 1746 (C=O), 2955, 2872, 1599, 1589, 1492, 1418, 1370, 1294, 1227, 1154, 1070,

  • 3

    1061, 940, 856, 756, 697; 1H NMR (400 MHz, CDCl3): δ 0.95 (3H, t, J = 7.4 Hz, N(CH2)3CH3), 1.36 (2H,

    sextet, J = 7.4 Hz, NCH2CH2CH2CH3), 1.37 and 1.46 (2 x 3H, 2 x s, Cquat(CH3)2), 1.57-1.72 (2H, m,

    NCH2CH2CH2CH3), 3.29 (1H, d x d x d, J = 13.8, 7.7, 6.0 Hz, N(HCH)), 3.50 (1H, d x t, J = 13.8, 7.4 Hz,

    N(HCH)), 3.68 (1H, d x d, J = 8.9, 6.3 Hz, O(HCH)), 3.82 (1H, d x d, J = 9.0, 5.0 Hz, NCH), 4.18 (1H, d x d,

    J = 8.9, 6.3 Hz, O(HCH)), 4.42 (1H, d x t, J = 9.0, 6.3 Hz, OCHCH2), 5.18 (1H, d, J = 5.0 Hz, PhOCH), 6.99-

    7.03 (1H, m, CHarom), 7.06-7.09 (2H, m, CHarom), 7.26-7.31 (2H, m, CHarom); 13C NMR (100 MHz, CDCl3):

    δ 13.6 (N(CH2)3CH3), 20.1 (NCH2CH2CH2CH3), 25.1 and 26.9 (Cquat(CH3)2), 29.4 (NCH2CH2CH2CH3), 41.3

    (NCH2), 60.4 (NCH), 67.0 (OCH2), 77.2 (OCHCH2), 79.8 (PhOCH), 109.7 (Cquat(CH3)2), 115.8 (2 x HCarom),

    122.5 (HCarom), 129.7 (2 x HCarom), 157.5 (Cquat,arom), 165.9 (C=O); MS (70 eV): m/z (%) = 320 (M+ + H,

    100); HRMS (ESI): m/z [M + H]+ calcd for C18H26NO4: 320.1862, found: 320.1863; Anal. calcd. (%) for

    C18H25NO4: C 67.69, H 7.89, N 4.39, found: C 66.53, H 8.48, N 4.25.

    Synthesis of 4-(hydroxymethyl)azetidin-2-ones 7a–c

    4-(Hydroxymethyl)azetidin-2-ones 7a–c were prepared in three steps from the corresponding 4-[(4S)-

    2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-ones 6a–c according to a literature procedure.47,48

    (3R,4S)-4-Hydroxymethyl-1-isopropyl-3-methoxyazetidin-2-one 7a

    Yellow oil; yield: 0.40 g (82%); Rf 0.32 (hexane/EtOAc 2/1); [α]D +181.8 (c 0.10 in CHCl3); IR (νmax/cm-1):

    3425 (OH), 1726 (C=O), 2934, 1461, 1350, 1213, 1140, 1034, 920, 792, 731, 646; 1H NMR (400 MHz,

    CDCl3): δ 1.25 and 1.26 (2 x 3H, 2 x d, J = 6.8 Hz, NCH(CH3)2), 2.36-2.39 (1H, m, OH), 3.62 (3H, s,

    OCH3), 3.80-3.94 (4H, m, NCHCH2 and NCH(CH3)2), 4.47 (1H, d, J = 4.7 Hz, OCH); 13C NMR (100 MHz,

    CDCl3): δ 20.0 and 21.6 (NCH(CH3)2), 44.1 (NCH(CH3)2), 56.7 (NCHCH2), 59.3 (OCH3), 61.1 (NCHCH2),

    83.4 (OCH), 166.5 (C=O); MS (70 eV): m/z (%) = 174 (M+ + H, 61); HRMS (ESI): m/z [M + H]+ calcd for

    C8H16NO3: 174.1130, found: 174.1130.

    (3R,4S)-3-Benzyloxy-4-hydroxymethyl-1-isopropylazetidin-2-one 7b

    Colorless oil; yield: 1.20 g (96%); Rf 0.11 (hexane/EtOAc 2/1); [α]D +138.7 (c 2.5 in CHCl3); IR (νmax/cm-

    1): 3417 (OH), 1724 (C=O), 2931, 1402, 1340, 1216, 1037, 1023, 738, 698; 1H NMR (300 MHz, CDCl3): δ

    1.25 (6H, d, J = 6.6 Hz, NCH(CH3)2), 2.48 (1H, br s, OH), 3.74-3.94 (4H, m, NCHCH2 and NCH(CH3)2),

    4.65 (1H, d, J = 4.9 Hz, CHO), 4.70 and 4.95 (2 x 1H, 2 x d, J = 11.6 Hz, O(HCH)Ph), 7.36-7.49 (5H, m,

    CHarom); 13C NMR (75 MHz, CDCl3): δ 20.1 and 21.7 (NCH(CH3)2), 44.0 (NCH(CH3)2), 56.8 (NCHCH2), 61.0

    (NCHCH2), 73.5 (OCH2Ph), 81.0 (CHO), 128.2, 128.4 and 128.7 (5 x HCarom), 136.7 (Carom,quat), 166.6

    (C=O); MS (70 eV): m/z (%) = 250 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C14H20NO3:

    250.1443, found: 250.1445.

  • 4

    (3R,4S)-1-Butyl-4-hydroxymethyl-3-phenoxyazetidin-2-one 7c

    Yellow oil; yield: 1.12 g (91%); Rf 0.44 (hexane/EtOAc 1/1); [α]D +53.3 (c 0.17 in CHCl3); IR (νmax/cm-1):

    3424 (OH), 1739 (C=O), 2932, 2874, 1598, 1494, 1415, 1353, 1233, 1174, 1108, 1047, 961, 886, 842,

    753, 734, 691; 1H NMR (400 MHz, CDCl3): δ 0.95 (3H, t, J = 7.4 Hz, N(CH2)3CH3), 1.38 (2H, sextet, J = 7.4

    Hz, NCH2CH2CH2CH3), 1.51-1.69 (2H, m, NCH2CH2CH2CH3), 1.97 (1H, br s, OH), 3.20 and 3.45 (2 x 1H, 2

    x (d x d x d), J = 14.4, 8.0, 7.8, 6.5, 6.1 Hz, N(HCH)), 3.98-3.99 (3H, m, NCHCH2OH), 5.28 (1H, d, J = 4.5

    Hz, OCH), 7.03-7.11 (3H, m, CHarom), 7.29-7.34 (2H, m, CHarom); 13C NMR (100 MHz, CDCl3): δ 13.6

    (N(CH2)3CH3), 20.3 (NCH2CH2CH2CH3), 29.8 (NCH2CH2CH2CH3), 40.8 (NCH2), 58.3 (NCH), 60.3 (CH2OH),

    80.6 (OCH), 115.7 (2 x HCarom), 122.7 (HCarom), 129.7 (2 x HCarom), 157.3 (Cquat,arom), 165.8 (C=O); MS (70

    eV): m/z (%) = 250 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C14H20NO3: 250.1443, found:

    250.1446.

    Synthesis of 4-(mesyloxymethyl)azetidin-2-ones 8a–b

    As a representative example, the synthesis of (3R,4S)-1-isopropyl-4-mesyloxymethyl-3-

    methoxyazetidin-2-one 8a is described. A solution of triethylamine (4.05 g, 40 mmol, 4 equiv) in dry

    THF (16 mL) was added dropwise to a solution of (3R,4S)-4-hydroxymethyl-1-isopropyl-3-

    methoxyazetidin-2-one 7a (1.74 g, 10 mmol, 1 equiv) in dry THF (30 mL) at room temperature. The

    resulting mixture was cooled to 0 °C and a solution of methanesulfonyl chloride (1.72 g, 15 mmol, 1.5

    equiv) in dry THF (7 mL) was added dropwise, after which the mixture was stirred for 1 hour at 0 °C.

    Afterward, the reaction mixture was washed with brine (2 x 25 mL) and a saturated NaHCO3 solution

    (2 x 25 mL). The combined aqueous phases were extracted again with EtOAc (40 mL). Drying of the

    combined organic phases with MgSO4, filtration of the drying agent, and removal of the solvent in

    vacuo afforded crude (3R,4S)-1-isopropyl-4-mesyloxymethyl-3-methoxyazetidin-2-one 8a, which was

    purified in 95% (2.39 g) yield by column chromatography on silica gel (hexane/EtOAc 1/1).

    (3R,4S)-1-Isopropyl-4-mesyloxymethyl-3-methoxyazetidin-2-one 8a

    Yellow oil; yield: 2.39 g (95%); Rf 0.11 (hexane/EtOAc 1/1); [α]D +53.7 (c 0.66 in CHCl3); IR (νmax/cm-1):

    1742 (C=O), 1391, 1351, 1216, 1172, 1142, 1032, 961, 824, 798; 1H NMR (400 MHz, CDCl3): δ 1.25 and

    1.28 (2 x 3H, 2 x d, J = 6.8 Hz, NCH(CH3)2), 3.06 (3H, s, SCH3), 3.55 (3H, s, OCH3), 3.89 (1H, septet, J =

    6.8 Hz, NCH(CH3)2), 4.05 (1H, d x d x d, J = 6.4, 5.0, 4.9 Hz, NCHCH2), 4.32 and 4.46 (2 x 1H, 2 x (d x d),

    J = 10.9, 6.4, 5.0 Hz, NCH(HCH), 4.50 (1H, d, J = 4.9 Hz, OCH); 13C NMR (100 MHz, CDCl3): δ 19.9 and

    21.8 (NCH(CH3)2), 37.4 (SCH3), 44.4 (NCH(CH3)2), 55.1 (NCHCH2), 59.4 (OCH3), 68.5 (NCHCH2), 82.7

    (OCH), 166.4 (C=O); MS (70 eV): m/z (%) = 252 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for

    C9H18NO5S: 252.0906, found: 252.0900.

  • 5

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(mesyloxymethyl)azetidin-2-one 8b

    Light-yellow oil; yield: 1.12 g (84%); Rf 0.10 (hexane/EtOAc 2/1); [α]D +62.8 (c 1.9 in CHCl3); IR

    (νmax/cm-1): 1727 (C=O), 2941, 1348, 1168, 1149, 976, 958, 823, 697; 1H NMR (300 MHz, CDCl3): δ 1.25

    and 1.27 (2 x 3H, 2 x d, J = 6.7 Hz, NCH(CH3)2), 2.95 (3H, s, SCH3), 3.90 (1H, septet, J = 6.7 Hz,

    NCH(CH3)2), 4.04 (1H, d x d x d, J = 6.3, 5.1, 5.0 Hz, NCHCH2), 4.32 and 4.41 (2 x 1H, 2 x (d x d), J =

    10.9, 6.3, 5.0 Hz, NCH(HCH), 4.67 (1H, d, J = 11.5 Hz, O(HCH)Ph, 4.69 (1H, d, J = 5.1 Hz, CHO), 4.86

    (1H, d, J = 11.5 Hz, O(HCH)Ph), 7.31-7.38 (5H, m, CHarom); 13C NMR (75 MHz, CDCl3): δ 20.0 and 21.8

    (NCH(CH3)2), 37.3 (SCH3), 44.5 (NCH(CH3)2), 55.1 (NCHCH2), 68.7 (NCHCH2), 73.5 (OCH2Ph), 80.5

    (CHO), 128.3, 128.4 and 128.7 (5 x HCarom), 136.7 (Carom,quat), 166.6 (C=O); MS (70 eV): m/z (%) = 328

    (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C15H22NO5S: 328.1219, found: 328.1223.

    Synthesis of 4-(tosyloxymethyl)azetidin-2-ones 9a–c

    A as a representative example, the synthesis of (3R,4S)-1-butyl-3-phenoxy-4-

    (tosyloxymethyl)azetidin-2-one 9c is described. A solution of triethylamine (2.02 g, 20 mmol, 4 equiv)

    and 4-dimethylaminopyridine (0.06 g, 0.5 mmol, 0.1 equiv) in dry CH2Cl2 (20 mL) was added dropwise

    to an ice-cooled solution of (3R,4S)-1-butyl-4-hydroxymethyl-3-phenoxyazetidin-2-one 7c (1.25 g, 5

    mmol, 1 equiv) in dry CH2Cl2 (15 mL). Subsequently, a solution of p-toluenesulfonyl chloride (1.43 g,

    7.5 mmol, 1.5 equiv) in dry CH2Cl2 (10 mL) was added dropwise, after which the mixture was stirred

    for 40-48 hours under reflux. Afterward, the reaction mixture was washed with brine (2 x 25 mL) and

    a saturated NaHCO3 solution (2 x 25 mL). The combined aqueous phases were extracted again with

    EtOAc (2 x 30 mL). Drying of the combined organic phases with MgSO4, filtration of the drying agent,

    and removal of the solvent in vacuo afforded crude (3R,4S)-1-butyl-3-phenoxy-4-

    (tosyloxymethyl)azetidin-2-one 9c, which was purified in 62% (1.27 g) yield by column

    chromatography on silica gel (hexane/EtOAc 3/1).

    (3R,4S)-1-Isopropyl-3-methoxy-4-(tosyloxymethyl)azetidin-2-one 9a

    Yellow oil; yield: 1.32 g (67%); Rf 0.28 (hexane/EtOAc 1/1); [α]D +329.5 (c 0.23 in CHCl3); IR (νmax/cm-1):

    1748 (C=O), 2936, 1598, 1454, 1360, 1175, 1096, 1032, 974, 815, 767, 731, 666; 1H NMR (400 MHz,

    CDCl3): δ 1.17 and 1.22 (2 x 3H, 2 x d, J = 6.8 Hz, NCH(CH3)2), 2.47 (3H, s, PhCH3), 3.45 (3H, s, OCH3),

    3.82 (1H, septet, J = 6.8 Hz, NCH(CH3)2), 3.99 (1H, d x d x d, J = 7.2, 4.8, 4.5 Hz, NCHCH2), 4.09 and

    4,24 (2 x 1H, 2 x (d x d), J = 10.7, 7.2, 4.5 Hz, NCH(HCH)), 4.43 (1H, d, J = 4.8 Hz, OCH), 7.38 (2H, d x d,

    J = 8.5, 0.6 Hz, 2 x CHarom), 7.81 (2H, d, J = 8.5 Hz, 2 x CHarom); 13C NMR (100 MHz, CDCl3): δ 19.8 and

    21.6 (NCH(CH3)2), 21.7 (PhCH3), 44.4 (NCH(CH3)2), 55.0 (NCHCH2), 59.4 (OCH3), 69.3 (CH2O), 82.6

    (OCH), 128.0 and 130.0 (4 x HCarom), 132.3 (CH3Cquat,arom), 145.4 (SCquat,arom), 166.3 (C=O); MS (70 eV):

  • 6

    m/z (%) = 328 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C15H22NO5S: 328.1219, found:

    328.1217.

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(tosyloxymethyl)azetidin-2-one 9b

    White crystals; yield: 0.96 g (60%); Rf 0.16 (hexane/EtOAc 2/1); mp 86 °C; [α]D +31.8 (c 2.7 in CHCl3);

    IR (νmax/cm-1): 1755 (C=O), 2918, 1364, 1350, 1175, 1148, 1015, 972, 850, 763, 749, 702, 663; 1H NMR

    (300 MHz, CDCl3): δ 1.17 and 1.21 (2 x 3H, 2 x d, J = 6.7 Hz, NCH(CH3)2), 2.45 (3H, s, CquatCH3), 3.82

    (1H, septet, J = 6.7 Hz, NCH(CH3)2), 3.97 (1H, d x d x d, J = 7.1, 4.7, 4.5 Hz, NCHCH2), 4.11 and 4.23 (2 x

    1H, 2 x (d x d), J = 10.5, 7.1, 4.5 Hz, NCH(HCH)), 4.60 (1H, d, J = 12.1 Hz, O(HCH)Ph), 4.61 (1H, d, J = 4.7

    Hz, CHO), 4.75 (1H, d, J = 12.1 Hz, O(HCH)Ph), 7.27-7.33 (7H, m, CHarom), 7.76 (2H, J = 8.2 Hz, CHarom);

    13C NMR (75 MHz, CDCl3): δ 19.9 (CH3CHCH3), 21.7 and 21.8 (CH3CHCH3 and CquatCH3), 44.6

    (NCH(CH3)2), 55.2 (NCHCH2), 69.5 (NCHCH2), 73.3 (OCH2Ph), 80.4 (CHO), 128.0, 128.1, 128.3, 128.6

    and 130.1 (9 x HCarom), 132.5 (SCarom,quat), 136.7 (CH2Carom,quat), 145.4 (CH3Carom,quat), 166.5 (C=O); MS

    (70 eV): m/z (%) = 404 (M+ + H, 100); Anal. calcd. (%) for C21H25NO5S: C 62.51, H 6.25, N 3.47, found: C

    62.70, H 6.54, N 3.51.

    (3R,4S)-1-Butyl-3-phenoxy-4-(tosyloxymethyl)azetidin-2-one 9c

    Yellow crystals; yield: 1.27 g (62%); Rf 0.27 (hexane/EtOAc 3/1); mp 68 °C; [α]D +23.0 (c 0.33 in CHCl3);

    IR (νmax/cm-1): 1746 (C=O), 2949, 2873, 1598, 1492, 1418, 1355, 1292, 1238, 1228, 1189, 1171, 968,

    896, 850, 829, 812, 764, 735, 699, 656; 1H NMR (400 MHz, CDCl3): δ 0.92 (3H, t, J = 7.4 Hz,

    N(CH2)3CH3), 1.31 (2H, sextet, J = 7.4 Hz, NCH2CH2CH2CH3), 1.47-1.64 (2H, m, NCH2CH2CH2CH3), 2.46

    (3H, s, ArCH3), 3.11 and 3.40 (2 x 1H, 2 x (d x d x d), J = 14.0, 8.1, 8.0, 7.2, 5.9 Hz, N(HCH)), 4.18 (1H, d

    x d x d, J = 7.7, 4.6, 3.5 Hz, NCH), 4.24 and 4.37 (2 x 1H, 2 x (d x d), J = 10.4, 7.7, 3.5 Hz, (HCH)O), 5.24

    (1H, d, J = 4.6 Hz, OCH), 6.91-7.05 (3H, m, CHarom), 7.28-7.34 (4H, m, CHarom), 7.71-7.73 (2H, m,

    CHarom); 13C NMR (100 MHz, CDCl3): δ 13.5 (N(CH2)3CH3), 20.1 (NCH2CH2CH2CH3), 21.6 (ArCH3), 29.5

    (NCH2CH2CH2CH3), 41.2 (NCH2), 56.1 (NCH), 69.0 (CH2O), 80.0 (OCH), 115.5 (2 x HCarom), 122.5

    (HCarom), 127.9 (2 x HCarom), 129.6 (2 x HCarom), 130.0 (2 x HCarom), 132.2 (CH3Cquat,arom), 145.3 and 157.2

    (OCquat,arom and SCquat,arom), 165.3 (C=O). MS (70 eV): m/z (%) = 404 (M+ + H, 10), 421 (M+ + NH4, 100);

    HRMS (ESI): m/z [M + H]+ calcd for C21H26NO5S: 404.1532, found: 404.1539; Anal. calcd. (%) for

    C21H25NO5S: C 62.51, H 6.25, N 3.47, found: C 62.85, H 6.46, N 3.37.

    Synthesis of 4-(iodomethyl)azetidin-2-ones 10a–c

    As a representative example, the synthesis of (3R,4R)-4-iodomethyl-1-isopropyl-3-methoxyazetidin-

    2-one 10a is described. To a solution of (3R,4S)-1-isopropyl-3-methoxy-4-(tosyloxymethyl)azetidin-2-

    one 9a (0.33 g, 1 mmol, 1 equiv) in dry acetone (10 mL), sodium iodide (0.60 g, 4 mmol, 4 equiv) was

  • 7

    added. After a reflux period of 40 hours, the solvent was evaporated, after which water (10 mL) was

    added and the mixture was extracted with EtOAc (3 × 15 mL). Drying of the combined organic phases

    with MgSO4, filtration of the drying agent, and removal of the solvent in vacuo afforded crude

    (3R,4R)-4-iodomethyl-1-isopropyl-3-methoxyazetidin-2-one 10a, which was purified in 80% (0.23 g)

    yield by column chromatography on silica gel (hexane/EtOAc 3/1).

    (3R,4R)-4-Iodomethyl-1-isopropyl-3-methoxyazetidin-2-one 10a

    Yellow oil; yield: 0.23 g (80%); Rf 0.24 (hexane/EtOAc 3/1); [α]D +138.1 (c 0.39 in CHCl3); IR (νmax/cm-1):

    1740 (C=O), 2971, 2932, 1462, 1389, 1340, 1236, 1212, 1170, 1143, 1033, 861, 730; 1H NMR (400

    MHz, CDCl3): δ 1.27 and 1.30 (2 x 3H, 2 x d, J = 6.8 Hz, NCH(CH3)2), 3.28 and 3.31 (2 x 1H, 2 x (d x d), J

    = 10.0, 8.4, 4.8 Hz, (HCH)I), 3.62 (3H, s, CH3O), 3.86 (1H, septet, J = 6.8 Hz, NCH(CH3)2), 4.11 (1H, d x d

    x d, J = 8.4, 4.8, 4.7 Hz, NCHCH2), 4.41 (1H, d, J = 4.7 Hz, OCH); 13C NMR (100 MHz, CDCl3): δ -1.5

    (CH2I), 18.2 and 20.2 (NCH(CH3)2), 42.4 (NCH(CH3)2), 56.7 (NCHCH2), 58.5 (CH3O), 81.6 (OCH), 164.7

    (C=O); MS (70 eV): m/z (%) = 284 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C8H15NO2I:

    284.0147, found: 284.0151.

    (3R,4R)-3-Benzyloxy-4-iodomethyl-1-isopropylazetidin-2-one 10b

    Light-yellow crystals; yield: 0.31 g (88%); Rf 0.22 (hexane/EtOAc 3/1); mp 60 °C; [α]D +162.9 (c 1.8 in

    CHCl3); IR (νmax/cm-1): 1724 (C=O), 2970, 1340, 1227, 1180, 1158, 1049, 735, 696, 641; 1H NMR (300

    MHz, CDCl3): δ 1.26 and 1.28 (2 x 3H, 2 x d, J = 7.0 Hz, NCH(CH3)2), 3.32 (2H, d, J = 5.8 Hz, CH2I), 3.87

    (1H, septet, J = 7.0 Hz, NCH(CH3)2), 4.10 (1H, q, J = 5.8 Hz, NCHCH2), 4.59 (1H, d, J = 5.8 Hz, CHO), 4.81

    and 4.87 (2 x 1H, 2 x d, J = 11.9 Hz, (HCH)O), 7.30-7.44 (5H, m, CHarom); 13C NMR (75 MHz, CDCl3): δ 0.9

    (CH2I), 20.3 and 22.2 (NCH(CH3)2), 44.5 (NCH(CH3)2), 58.7 (NCHCH2), 73.9 (CH2O), 81.3 (CHO), 128.0,

    128.1 and 128.5 (5 x HCarom), 137.2 (Carom,quat), 166.8 (C=O); MS (70 eV): m/z (%) = 360 (M+ + H, 100);

    Anal. calcd. (%) for C14H18NO2I: C 46.81, H 5.05, N 3.90, found: C 47.37, H 5.14, N 3.86.

    (3R,4R)-1-Butyl-4-iodomethyl-3-phenoxyazetidin-2-one 10c

    Yellow oil; yield: 0.31 g (69%); Rf 0.21 (hexane/EtOAc 4/1); [α]D +281.2 (c 0.37 in CHCl3); IR (νmax/cm-1):

    1751 (C=O), 2957, 2929, 1597, 1590, 1493, 1402, 1347, 1233, 1171, 1045, 931, 884, 862, 752, 690; 1H

    NMR (400 MHz, CDCl3): δ 0.96 (3H, t, J = 7.4 Hz, N(CH2)3CH3), 1.37 (2H, sextet, J = 7.4 Hz,

    NCH2CH2CH2CH3), 1.58-1.73 (2H, m, NCH2CH2CH2CH3), 3.32 (1H, d x d x d, J = 14.0, 8.1, 5.7 Hz,

    N(HCH)), 3.35 and 3.39 (2 x 1H, 2 x (d x d), J = 10.5, 6.7, 6.5 Hz, (HCH)I), 3.47 (1H, d x d x d, J = 14.0,

    8.3, 7.2 Hz, N(HCH)), 4.28 (1H, d x d x d, J = 6.7, 6.5, 4.7 Hz, NCH), 5.21 (1H, d, J = 4.7 Hz, OCH), 7.01-

    7.11 (3H, m, CHarom), 7.29-7.34 (2H, m, CHarom); 13C NMR (100 MHz, CDCl3): δ -0.1 (CH2I), 13.7

    (N(CH2)3CH3), 20.2 (NCH2CH2CH2CH3), 29.9 (NCH2CH2CH2CH3), 40.6 (NCH2), 59.5 (NCH), 81.1 (OCH),

  • 8

    115.9 (2 x HCarom), 122.5 (HCarom), 129.7 (2 x HCarom), 157.5 (Cquat,arom), 165.8 (C=O); MS (70 eV): m/z

    (%) = 360 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for C14H19NO2I: 360.0460, found: 360.0467.

    Synthesis of 4-(cyanomethyl)azetidin-2-ones 11a–c

    As a representative example, the synthesis of (3R,4S)-4-cyanomethyl-1-isopropyl-3-methoxyazetidin-

    2-one 11a is described. To a solution of (3R,4R)-4-iodomethyl-1-isopropyl-3-methoxyazetidin-2-one

    10a (0.28 g, 1 mmol, 1 equiv) in dry DMF (3 mL), crushed sodium cyanide (0.12 g, 2.5 mmol, 2.5

    equiv) was added. After stirring for 72 hours at room temperature, brine was added (30 mL) and the

    resulting mixture was extracted with Et2O (2 × 20 mL) and EtOAc (20 mL). Drying of the combined

    organic phases with MgSO4, filtration of the drying agent, and removal of the solvent in vacuo

    afforded crude (3R,4S)-4-cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a, which was purified

    in 62% (0.11 g) yield by column chromatography on silica gel (hexane/EtOAc 4/1).

    (3R,4S)-4-Cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a

    Yellow oil; yield: 0.11 g (62%); Rf 0.12 (hexane/EtOAc 4/1); [α]D +50.6 (c 0.20 in CHCl3); IR (νmax/cm-1):

    2251 (C≡N), 1743 (C=O), 2936, 2840, 1672, 1465, 1394, 1370, 1344, 1250, 1214, 1140, 1089, 1034,

    729, 647; 1H NMR (400 MHz, CDCl3): δ 1.28 and 1.31 (2 x 3H, 2 x d, J = 6.8 Hz, NCH(CH3)2), 2.64 and

    2.74 (2 x 1H, 2 x (d x d), J = 17.1, 6.1, 5.8 Hz, (HCH)CN)), 3.60 (3H, s, CH3O), 3.88 (1H, septet, J = 6.8

    Hz, NCH(CH3)2), 4.05 (1H, d x d x d, J = 6.1, 5.8, 5.3 Hz, NCHCH2), 4.48 (1H, d, J = 5.3 Hz, OCH); 13C NMR

    (100 MHz, CDCl3): δ 18.9 (CH2CN), 20.1 and 21.8 (NCH(CH3)2), 44.5 (NCH(CH3)2), 53.1 (NCHCH2), 59.6

    (CH3O), 82.7 (OCH), 117.1 (CN), 166.0 (C=O); MS (70 eV): m/z (%) = 183 (M+ + H, 65), 200 (M+ + NH4,

    100); HRMS (ESI): m/z [M + H]+ calcd for C9H15N2O2: 183.1134, found: 183.1134.

    (3R,4S)-3-Benzyloxy-4-cyanomethyl-1-isopropylazetidin-2-one 11b

    White crystals; yield: 0.26 g (51%); Rf 0.05 (hexane/EtOAc 9/1); mp

  • 9

    (3R,4S)-1-Butyl-4-cyanomethyl-3-phenoxyazetidin-2-one 11c

    Yellow crystals; yield: 0.12 g (53%); Rf 0.15 (hexane/EtOAc 4/1); mp 55 °C; [α]D +67.2 (c 0.23 in CHCl3);

    IR (νmax/cm-1): 2252 (C≡N), 1753 (C=O), 2931, 1591, 1494, 1458, 1407, 1351, 913, 754, 731, 691; 1H

    NMR (400 MHz, CDCl3): δ 0.97 (3H, t, J = 7.4 Hz, N(CH2)3CH3), 1.39 (2H, sextet, J = 7.4 Hz,

    NCH2CH2CH2CH3), 1.57-1.71 (2H, m, NCH2CH2CH2CH3), 2.75 and 2.81 (2 x 1H, 2 x (d x d), J = 17.2, 7.3,

    5.2 Hz, (HCH)CN), 3.26 and 3.52 (2 x 1H, 2 x (d x d x d), J = 14.1, 8.2, 8.1, 7.1, 5.9 Hz, N(HCH)), 4.23

    (1H, d x d x d, J = 7.3, 5.2, 4.9 Hz, NCH), 5.30 (1H, d, J = 4.9 Hz, CHO), 7.04-7.08 (3H, m, CHarom), 7.30-

    7.34 (2H, m, CHarom); 13C NMR (100 MHz, CDCl3): δ 13.6 (N(CH2)3CH3), 18.4 (CH2CN), 20.2

    (NCH2CH2CH2CH3), 29.7 (NCH2CH2CH2CH3), 40.9 (NCH2), 54.2 (NCH), 80.3 (CHO), 115.7 (2 x HCarom),

    116.8 (CN), 122.9 (HCarom), 129.8 (2 x HCarom), 156.9 (Cquat,arom), 164.9 (C=O); MS (70 eV): m/z (%) = 259

    (M+ + H, 12), 276 (M+ + NH4, 100); HRMS (ESI): m/z [M + H]+ calcd for C15H19N2O2: 259.1447, found:

    259.1445.

    Synthesis of 4-(carboxymethyl)azetidin-2-ones 12a–c

    As a representative example, the synthesis of (3R,4S)-4-carboxymethyl-1-isopropyl-3-

    methoxyazetidin-2-one 12a is described.

    Method A: 1 mg/mL nitrilase NIT-P1-12168 was added to 20 mL of a 20 mM solution of (3R,4S)-4-

    cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a in a K3PO4-dithiothreitol-

    ethylenediaminetetraacetic acid buffer (50 mM/20 mM/1 mM, pH = 7.5) (95%) and MeOH (5%), and

    the resulting mixture was agitated in a thermomixer (30 °C, 200 rpm). Every 72 h, 1 mg/mL nitrilase

    NIT-P1-121 was added until a concentration of 4 mg/mL was reached. After four weeks of incubation,

    60% conversion toward (3R,4S)-4-carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a was

    attained (ratio determined on the basis of LC-MS analysis). The enzyme was inactivated by heat

    treatment (95 °C, 10 min – 0 °C, 10 min) and removed by centrifugation (14000 rpm, 10 min), after

    which the liquid phase was evaporated in vacuo. Removal of the buffer salts was achieved by

    filtration after resolvation in MeOH and EtOAc.

    Finally, pure (3R,4S)-4-carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a was obtained in 16%

    (7 mg) yield after separation from the starting material 11a by preparative HPLC.

    Method B: 1 mg/mL nitrilase NIT-P1-12168 was added to 20 mL of a 20 mM solution of (3R,4S)-4-

    cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a in a K3PO4-dithiothreitol-

    ethylenediaminetetraacetic acid buffer (50 mM/20 mM/1 mM, pH = 7.5) (95%) and MeOH (5%), and

    the resulting mixture was agitated in a thermomixer (30 °C, 200 rpm). Every 72 h, 1 mg/mL nitrilase

    NIT-P1-121 was added until a concentration of 4 mg/mL was reached. After four weeks of incubation,

  • 10

    60% conversion toward (3R,4S)-4-carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a was

    attained (ratio determined on the basis of LC-MS analysis). The enzyme was inactivated by heat

    treatment (95 °C, 10 min – 0 °C, 10 min) and removed by centrifugation (14000 rpm, 10 min). In

    order to remove the unreacted starting material 11a, the crude reaction mixture was saturated with

    sodium chloride and extracted with EtOAc (3 x 30 mL). Subsequently, the mixture was acidified to pH

    4 by HCl, saturated with sodium chloride and extracted with EtOAc (3 x 30 mL). Drying of the latter

    combined organic phases with MgSO4, filtration of the drying agent, and removal of the solvent in

    vacuo afforded (3R,4S)-4-carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a in 43% (19 mg)

    yield.

    (3R,4S)-4-Carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a

    Spectral data based on 1H NMR and 13C NMR analysis of the crude reaction mixture (purity ~85%

    according to 1H NMR ). Yellow oil; yield: 19 mg (43%); Rf 0.18 (hexane/EtOAc 2/1); [α]D +92.9 (c 0.20

    in CHCl3); IR (νmax/cm-1): 3415 (OH), 1732 (NC=O), 1648 (OC=O), 1403, 1369, 1348, 1242, 1209, 1177,

    1037; 1H NMR (400 MHz, CDCl3): δ 1.23 and 1.27 (2 x 3H, 2 x d, J = 6.7 Hz, NCH(CH3)2), 2.75 and 2.82

    (2 x 1H, 2 x (d x d), J = 17.2, 7.7, 5.5 Hz, (HCH)COOH), 3.55 (3H, s, OCH3), 3.86 (1H, septet, J = 6.7 Hz,

    NCH(CH3)2), 4.21 (1H, d x d x d, J = 7.7, 5.5, 5.0 Hz, NCHCH2), 4.48 (1H, d, J = 5.0 Hz, OCH); 13C NMR

    (100 MHz, CDCl3): δ 20.1 and 21.6 (NCH(CH3)2),

    34.2 (CH2COOH), 44.1 (NCH(CH3)2), 52.9 (NCHCH2),

    59.4 (OCH3), 82.9 (OCH), 166.8 (NCO), 175.2 (COOH); MS (70 eV): m/z (%) = 202 (M+ + H, 72); HRMS

    (ESI): m/z [M + H]+ calcd for C9H16NO4: 202.1079, found: 202.1086.

    (3R,4S)-3-Benzyloxy-4-carboxymethyl-1-isopropylazetidin-2-one 12b

    White crystals; yield: 40 mg (74%); Rf 0.08 (hexane/EtOAc 2/1); mp 94 °C; [α]D +60.5 (c 0.23 in CHCl3);

    IR (νmax/cm-1): 3401 (OH), 1726 (NC=O), 1693 (OC=O), 1242, 1196, 1175, 1140, 1031, 1020, 996, 753,

    705; 1H NMR (400 MHz, CDCl3): δ 1.21 and 1.25 (2 x 3H, 2 x d, J = 6.7 Hz, NCH(CH3)2), 2.76 and 2.85 (2

    x 1H, 2 x (d x d), J = 17.3, 7.7, 5.4 Hz, (HCH)COOH), 3.84 (1H, septet, J = 6.7 Hz, NCH(CH3)2), 4.19 (1H,

    d x d x d, J = 7.7, 5.4, 4.9 Hz, NCHCH2), 4.65 (1H, d, J = 4.9 Hz, OCH), 4.66 and 4.83 (2 x 1H, 2 x d, J =

    12.3 Hz, O(HCH)), 7.27-7.35 (5H, m, CHarom); 13C NMR (100 MHz, CDCl3): δ 20.1 and 21.6 (NCH(CH3)2),

    34.5 (CH2COOH), 44.2 (NCH(CH3)2), 53.0 (NCHCH2), 73.1 (OCH2), 80.6 (OCH), 127.7, 127.9 and 128.4 (5

    x HCarom), 137.0 (Carom,quat), 166.9 (NCO), 175.7 (COOH); MS (70 eV): m/z (%) = 278 (M+ + H, 100);

    HRMS (ESI): m/z [M + H]+ calcd for C15H20NO4: 278.1392, found: 278.1387.

  • 11

    (3R,4S)-1-Butyl-4-carboxymethyl-3-phenoxyazetidin-2-one 12c

    White crystals; yield: 10 mg (12%); Rf 0.13 (hexane/EtOAc 2/1); [α]D +48.0 (c 0.13 in CHCl3); IR

    (νmax/cm-1): 3423 (OH), 1731 (NC=O), 1700 (OC=O), 2957, 2871, 1589, 1486, 1433, 1354, 1307, 1212,

    1185, 1144, 1111, 1074, 1034, 841, 753, 692, 669; 1H NMR (400 MHz, CDCl3): δ 0.93 (3H, t, J = 7.3 Hz,

    N(CH2)3CH3), 1.34 (2H, sextet, J = 7.3 Hz, NCH2CH2CH2CH3), 1.49-1.63 (2H, m, NCH2CH2CH2CH3), 2.79

    and 2.87 (2 x 1H, 2 x (d x d), J = 17.5, 6.9, 6.1 Hz, (HCH)COOH), 3.08 and 3.43 (2 x 1H, 2 x (d x d x d), J

    = 14.1, 7.8, 7.5, 7.5, 6.1 Hz, N(HCH), 4.35 (1H, d x d x d, J = 6.9, 6.1, 4.8 Hz, NCH), 5.29 (1H, d, J = 4.8

    Hz, OCH), 6.99-7.05 (3H, m, CHarom), 7.27-7.30 (2H, m, CHarom); 13C NMR (100 MHz, CDCl3): δ 13.6

    (N(CH2)3CH3), 20.1 (NCH2CH2CH2CH3), 29.8 (NCH2CH2CH2CH3), 33.6 (CH2COOH), 40.6 (NCH2), 54.2

    (NCH), 80.3 (OCH), 115.6 (2 x HCarom), 122.4 (HCarom), 129.6 (2 x HCarom), 157.4 (Cquat,arom), 165.7

    (NC=O), 175.5 (COOH); MS (70 eV): m/z (%) = 278 (M+ + H, 100); HRMS (ESI): m/z [M + H]+ calcd for

    C15H20NO4: 278.1392, found: 278.1397.

  • 12

    (3R,4S)-1-Isopropyl-3-methoxy-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-one 6a

    NO

    MeOH H

    R S

    OO

    H

    S

  • 13

    (3R,4S)-1-Isopropyl-3-methoxy-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]azetidin-2-one 6a

    NO

    MeOH H

    R S

    OO

    H

    S

  • 14

    (3R,4S)-1-Butyl-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-phenoxyazetidin-2-one 6c

    NO

    H H

    R S

    OO

    O

    H

    S

  • 15

    (3R,4S)-1-Butyl-4-[(4S)-2,2-dimethyl-1,3-dioxolan-4-yl]-3-phenoxyazetidin-2-one 6c

    NO

    H H

    R S

    OO

    O

    H

    S

  • 16

    (3R,4S)-4-Hydroxymethyl-1-isopropyl-3-methoxyazetidin-2-one 7a

    N

    MeO

    O

    OH

    H H

    R S

  • 17

    (3R,4S)-4-Hydroxymethyl-1-isopropyl-3-methoxyazetidin-2-one 7a

    N

    MeO

    O

    OH

    H H

    R S

  • 18

    (3R,4S)-3-Benzyloxy-4-hydroxymethyl-1-isopropylazetidin-2-one 7b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    09.

    010

    .011

    .012

    .013

    .014

    .015

    .016

    .017

    .018

    .019

    .020

    .021

    .022

    .023

    .024

    .0

    X : parts per Million : 1H

    8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

    7.

    611

    7.

    453

    7.

    362

    7.

    323

    7.

    263

    5.

    302

    4.

    990

    4.

    950

    4.

    725

    4.

    686

    4.

    670

    4.

    653

    3.

    921

    3.

    899

    3.

    875

    3.

    860

    3.

    853

    3.

    838

    3.

    820

    3.

    807

    3.

    793

    3.

    782

    2.

    405

    2.

    383

    2.

    361

    1.

    580

    1.

    436

    1.

    379

    1.

    355

    1.

    289

    1.

    264

    1.

    242

    1.

    196

    1.

    179

    1.

    157

    1.

    051

    1.

    027

    0.

    004

    0.

    000

    1.30

    1.29

    0.89

    0.45

    0.23

    0.20

  • 19

    (3R,4S)-3-Benzyloxy-4-hydroxymethyl-1-isopropylazetidin-2-one 7b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    09.

    010

    .011

    .0

    X : parts per Million : 13C

    200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0

    167

    .516

    166

    .783

    136

    .781

    129

    .132

    128

    .673

    128

    .597

    128

    .338

    128

    .185

    128

    .032

    127

    .880

    126

    .994

    80.

    869

    79.

    785

    77.

    693

    77.

    266

    76.

    838

    73.

    434

    73.

    067

    71.

    708

    63.

    769

    60.

    944

    58.

    211

    57.

    036

    53.

    600

    45.

    936

    44.

    073

    31.

    003

    21.

    675

    21.

    308

    19.

    995

  • 20

    (3R,4S)-1-Butyl-4-hydroxymethyl-3-phenoxyazetidin-2-one 7c

    N

    O

    O

    OH

    H H

    R S

  • 21

    (3R,4S)-1-Butyl-4-hydroxymethyl-3-phenoxyazetidin-2-one 7c

    N

    O

    O

    OH

    H H

    R S

  • 22

    (3R,4S)-1-Isopropyl-4-mesyloxymethyl-3-methoxyazetidin-2-one 8a

  • 23

    (3R,4S)-1-Isopropyl-4-mesyloxymethyl-3-methoxyazetidin-2-one 8a

  • 24

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(mesyloxymethyl)azetidin-2-one 8b

    (M

    illi

    ons)

    010

    .020

    .030

    .0

    X : parts per Million : 1H

    11.0 10.0 9.0 8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0 -1.0 -2.0 -3.0

    7.

    402

    7.

    377

    7.

    373

    7.

    360

    7.

    349

    7.

    344

    7.

    334

    7.

    322

    7.

    305

    7.

    263

    4.

    884

    4.

    846

    4.

    701

    4.

    684

    4.

    646

    4.

    404

    4.

    388

    4.

    349

    4.

    329

    4.

    045

    4.

    025

    3.

    899

    3.

    181

    3.

    003

    2.

    950

    2.

    718

    1.

    337

    1.

    284

    1.

    273

    1.

    262

    1.

    252

    1.

    242

    4.95

    4.95

    4.95

    5.50

    6.60

    7.15

    6.60

    6.60

    7.15

    6.60

    6.60

    6.05

    1.00

    0.95

    0.47

    0.47

    0.31

    0.13

    0.12

    0.35

    m

  • 25

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(mesyloxymethyl)azetidin-2-one 8b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    0

    X : parts per Million : 13C

    220.0 210.0 200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0 -20.0

    166

    .584

    136

    .705

    128

    .673

    128

    .383

    128

    .261

    80.

    548

    77.

    647

    77.

    220

    76.

    792

    73.

    510

    68.

    746

    55.

    142

    44.

    546

    37.

    324

    29.

    767

    21.

    827

    19.

    980

  • 26

    (3R,4S)-1-Isopropyl-3-methoxy-4-(tosyloxymethyl)azetidin-2-one 9a

    N

    MeO

    O

    O

    H H

    R S

    S

    O

    O

  • 27

    (3R,4S)-1-Isopropyl-3-methoxy-4-(tosyloxymethyl)azetidin-2-one 9a

    N

    MeO

    O

    O

    H H

    R S

    S

    O

    O

  • 28

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(tosyloxymethyl)azetidin-2-one 9b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    0

    X : parts per Million : 1H

    8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

    7.

    776

    7.

    748

    7.

    629

    7.

    604

    7.

    558

    7.

    538

    7.

    464

    7.

    333

    7.

    309

    7.

    301

    7.

    274

    7.

    263

    4.

    769

    4.

    728

    4.

    619

    4.

    602

    4.

    578

    4.

    254

    4.

    239

    4.

    219

    4.

    205

    4.

    142

    4.

    119

    4.

    108

    4.

    084

    3.

    994

    3.

    979

    3.

    963

    3.

    956

    3.

    941

    3.

    840

    3.

    816

    3.

    794

    3.

    772

    2.

    448

    2.

    170

    1.

    253

    1.

    225

    1.

    203

    1.

    181

    1.

    159

    1.95

    1.03

    0.78

    0.450.

    47

    0.23

    1.56

    -4.8

    9m

    -36.

    68m

  • 29

    (3R,4S)-3-Benzyloxy-1-isopropyl-4-(tosyloxymethyl)azetidin-2-one 9b

    (M

    illi

    ons)

    00.

    10.

    20.

    30.

    40.

    50.

    60.

    70.

    80.

    91.

    01.

    11.

    21.

    31.

    41.

    51.

    61.

    71.

    81.

    92.

    02.

    12.

    22.

    32.

    42.

    52.

    6

    X : parts per Million : 13C

    200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0 -10.0

    166

    .493

    145

    .362

    136

    .689

    132

    .460

    130

    .093

    128

    .612

    128

    .277

    128

    .139

    128

    .048

    80.

    380

    77.

    556

    77.

    128

    76.

    716

    73.

    342

    69.

    540

    55.

    249

    44.

    592

    21.

    782

    21.

    720

    19.

    934

  • 30

    (3R,4S)-1-Butyl-3-phenoxy-4-(tosyloxymethyl)azetidin-2-one 9c

  • 31

    (3R,4S)-1-Butyl-3-phenoxy-4-(tosyloxymethyl)azetidin-2-one 9c

  • 32

    (3R,4R)-4-Iodomethyl-1-isopropyl-3-methoxyazetidin-2-one 10a

    N

    MeO

    O

    I

    H H

    R R

  • 33

    (3R,4R)-4-Iodomethyl-1-isopropyl-3-methoxyazetidin-2-one 10a

    N

    MeO

    O

    I

    H H

    R R

  • 34

    (3R,4R)-3-Benzyloxy-4-iodomethyl-1-isopropylazetidin-2-one 10b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    09.

    010

    .011

    .012

    .0

    X : parts per Million : 1H

    8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

    7.

    444

    7.

    420

    7.

    384

    7.

    362

    7.

    336

    7.

    323

    7.

    300

    7.

    278

    4.

    886

    4.

    847

    4.

    831

    4.

    791

    4.

    593

    4.

    578

    4.

    113

    4.

    093

    4.

    073

    3.

    890

    3.

    868

    3.

    844

    3.

    335

    3.

    313

    1.

    297

    1.

    271

    1.

    245

    0.

    000

    2.05

    1.26

    0.60

    0.27

    0.64

    0.27 0.

    30

  • 35

    (3R,4R)-3-Benzyloxy-4-iodomethyl-1-isopropylazetidin-2-one 10b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    0

    X : parts per Million : 13C

    200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

    166

    .783

    137

    .193

    128

    .521

    128

    .063

    127

    .956

    81.

    281

    77.

    617

    77.

    205

    76.

    777

    73.

    861

    58.

    700

    44.

    516

    29.

    797

    22.

    240

    20.

    316

    0.

    895

  • 36

    (3R,4R)-1-Butyl-4-iodomethyl-3-phenoxyazetidin-2-one 10c

    N

    O

    O

    I

    H H

    R R

  • 37

    (3R,4R)-1-Butyl-4-iodomethyl-3-phenoxyazetidin-2-one 10c

    N

    O

    O

    I

    H H

    R R

  • 38

    (3R,4S)-4-Cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a

    N

    MeO

    O

    CN

    H H

    R S

  • 39

    (3R,4S)-4-Cyanomethyl-1-isopropyl-3-methoxyazetidin-2-one 11a

    N

    MeO

    O

    CN

    H H

    R S

  • 40

    (3R,4S)-3-Benzyloxy-4-cyanomethyl-1-isopropylazetidin-2-one 11b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    09.

    0

    X : parts per Million : 1H

    7.0 6.0 5.0 4.0 3.0 2.0 1.0 0

    7.

    378

    7.

    373

    7.

    366

    7.

    338

    7.

    334

    7.

    314

    7.

    274

    7.

    131

    7.

    102

    4.

    886

    4.

    847

    4.

    730

    4.

    692

    4.

    664

    4.

    648

    4.

    038

    4.

    018

    4.

    001

    3.

    981

    3.

    910

    3.

    888

    3.

    866

    3.

    844

    3.

    820

    2.

    741

    2.

    719

    2.

    685

    2.

    663

    2.

    657

    2.

    637

    2.

    600

    2.

    580

    1.

    302

    1.

    278

    1.

    275

    1.

    253

    0.

    077

    -0.

    000

    2.40

    1.60

    0.71

    0.68

    0.32

    0.28

    0.27

  • 41

    (3R,4S)-3-Benzyloxy-4-cyanomethyl-1-isopropylazetidin-2-one 11b

    (M

    illi

    ons)

    01.

    02.

    03.

    04.

    05.

    06.

    07.

    08.

    0

    X : parts per Million : 13C

    200.0 190.0 180.0 170.0 160.0 150.0 140.0 130.0 120.0 110.0 100.0 90.0 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0

    166

    .187

    136

    .689

    129

    .880

    128

    .689

    128

    .368

    128

    .185

    127

    .086

    117

    .345

    80.

    548

    77.

    678

    77.

    266

    76.

    838

    73.

    571

    53.

    249

    44.

    592

    21.

    827

    20.

    148

    19.

    186

  • 42

    (3R,4S)-1-Butyl-4-cyanomethyl-3-phenoxyazetidin-2-one 11c

    N

    O

    O

    H H

    R S CN

  • 43

    (3R,4S)-1-Butyl-4-cyanomethyl-3-phenoxyazetidin-2-one 11c

    N

    O

    O

    H H

    R S CN

  • 44

    (3R,4S)-4-Carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a

  • 45

    (3R,4S)-4-Carboxymethyl-1-isopropyl-3-methoxyazetidin-2-one 12a

  • 46

    (3R,4S)-3-Benzyloxy-4-carboxymethyl-1-isopropylazetidin-2-one 12b

  • 47

    (3R,4S)-3-Benzyloxy-4-carboxymethyl-1-isopropylazetidin-2-one 12b

  • 48

    (3R,4S)-1-Butyl-4-carboxymethyl-3-phenoxyazetidin-2-one 12c

    N

    O

    O

    H H

    R S COOH

  • 49

    (3R,4S)-1-Butyl-4-carboxymethyl-3-phenoxyazetidin-2-one 12c

    N

    O

    O

    H H

    R S COOH